Chemotherapeutic agents that alkylate the O6 position of guanine in DNA such as Carmustine (Ishibashi, et al., J. Biol. Chem., 269: 7645-7650, 1994) 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, fotemustine, dacarbazine, streptozotocin, procarbazine, and temozolomide (TMZ) are used primarily to treat brain cancer, melanoma, lymphoma, and gastrointestinal cancers. The effectiveness of these agents, however, is limited by alkylguanyltransferase (AGT), a protein that repairs O6-alkylguanine adducts and is up-regulated in several tumors during progression (Ishibashi et al., Mutat. Res., 315: 199-212, 1994; Citron et al., Cancer Investig., 12: 605-610, 1994; Kokkinakis et al., Cancer Res., 57: 5360-5368, 1997).
Furthermore, selection of resistant AGT phenotypic populations after treatment with alkylating agents seems to be the reason for the recurrence of tumors of even a more resistant phenotype (Lage et al., J. Cancer Res. Clin. Oncol., 125: 156-165, 1999). Tumor resistance to DNA alkylation could be theoretically reversed with AGT inhibitors that react with and inactivate the protein. Despite considerable advances in this field, methodologies to sensitize tumors by depleting AGT and the selection of the appropriate chemotherapeutic agent to be combined with AGT depleting drugs are still under evaluation.
An additional important issue in combining DNA alkylating agents with AGT inhibitors is whether to include such inhibitors in the treatment of tumors with no or low AGT content, especially because such a combination limits the dose of the alkylating agent. Dose is important for several reasons, including the fact that the alkylating agent itself might quench low levels of AGT. A case in point is TMZ, which at a dose of 100 mg/kg eliminates all of the AGT activity in tumors having moderate AGT levels for a prolonged time period (Chinnasamy et al., Blood, 89: 1566-1573, 1997). In addition, the inverse correlation between AGT levels and effectiveness of BCNU against central nervous system tumors (Belanisch et al., Cancer Res., 56: 783-788, 1996; Jaeckle et al., J. Clin. Oncol., 16: 3310-3315, 1998) suggest that there may be no benefit in treating AGT-deficient tumors with AGT inhibitors. Determining the threshold of AGT activity that could be overcome by alkylating agents without the use of AGT inhibitors may be beneficial.
High AGT activity confers resistance to DNA alkyating agents (see above). A sensitive and fast turn-round AGT assay could be used to select patients with low AGT activities, and thus provide a better outcome for patients receiving DNA alkylating agents. A few assays have been developed but they are either not sensitive enough or so labor-intensive that they are not suitable for routine laboratory use (Wu et al., Cancer Res., 47: 6229, 1987; Gerson et al., J. clin. Invest., 76:2106, 1985; Kreklau et al., Nucleic Acid Res., 29:2558, 2001).
This invention provides an improved assay, which is simple and efficient, for alkylguanyltransferase.